Anti-ischemic compounds

ABSTRACT

The present invention is related to a compound of formula (I), salts and pro-drugs of the compound (I) and methods for treating and/or preventing partial or total ischemia, methods for treating and/or preventing pathologies associated with ischemia or with mitochondrial deficiencies.

RELATED APPLICATION DATA

This application is a National Phase application under 35 U.S.C. §371 ofInternational Application Number PCT/EP00/10085, filed Oct. 11, 2000which claims priority to European Application Number 99870212.0 filedOct. 15, 1999.

OBJECT OF THE INVENTION

The present invention relates to new therapeutic agents with aprotective effect on proteinic complexes of the inner mitochondrialmembrane and that can be used preferably for preventing and/or treatingpartial or total ischemia, pathologies associated with ischemia orassociated with mitochondrial deficiencies, or apoptosis.

INVENTION-BASED TECHNOLOGICAL BACKGROUND

Ischemia is an interruption of the blood irrigation of an organ or atissue through artery obliteration, compression or spasma. Theconsequences depend on the nature of the tissue being deprived ofoxygenated blood and the phenomenon duration. Total ischemia leads totissue necrosis in a period of time variable according to the oxygenneeds. Partial ischemia causes either a reduction of the organcapabilities (psychic slowing down, hepatic or kidney functioninsufficiency, etc.) or the incapability to modify its work at anystimulation, showing then signs of pain (angina pectoris crises,arteritis cramps). Ischemia is very deleterious for the nerve tissue. Itis first to be seen in reversible metabolic and functional troubles thatcan lead to an abnormal nervous activity, for example, an epilepsycrisis or some cerebral cortex ischemic lesions. After a few minutes ofischemia, irreversible lesions of the nervous tissue appear, accompaniedwith cellular body swelling followed by necrosis thereof. This is calledcerebral infarction.

It is known to use agonist molecules for alpha-adrenergic receptors andagonist molecules for alpha-pre-synaptic receptors for treatingischemia. Such compounds are used at the heart level for treating angor,which is a clinical expression of acute myocardial ischemia and theresult of a temporary imbalance between the myocardium oxygen demand andthe oxygen intake through circulation, leading possibly, in seriouscases, to a myocardial infarction.

STATE OF THE ART

The use of various plant extracts for treating and/or preventingischemia or pathologies associated with ischemia or an energy deficiencyis described in International Patent Application WO 98/51291. Thisdocument also describes the working mechanism of said molecules thatallow an inhibition of the activation cascade of the endothelial cellsinduced by hypoxia as well as the protective effect of such products onsome mitochondrial deficiencies, particularly with a protective effectof the mitochondria complexes I and III. This document gives abiochemical model making it possible to obtain in vitro a protectiveeffect from anti-ischemic compounds.

The Publication by Ukrainets I. et al. (Tetrahedron Letters Vol. 50, n°34, p. 10331-10338 (1994)) describes derivatives and the malonic acidsynthesis, particularly an ethyl malonic acid ester and symmetricaldiamylides.

Malonic acid derivatives and their use for delaying plant growth aredescribed in International Patent Application WO 87/05898.

The Publication by Venuti et al. (J. of Med. Chem. Vol. 31, p. 2145-2152(1988)) describes, by way of an intermediate compound, a malonic acidderivate involved in the synthesis of compounds being used as prodrugs.

The Publication by Oumar-Mahamat et al. (Tetrahedron Letters Vol. 30, n°3, p. 331-332 (1989)) describes cyclic malonic acid derivates.

Patent Application EP-A-0,099,091 describes hexahydrodioxypyrimidinederivates, the production method thereof and their use as antiviral,anti-bacterial and antitumor compounds.

Patent Application FR-2,539,412 describes 5-fluorouracyl derivates andtheir therapeutic use as carcinostatic agents.

The Publication by Holwood et al. (J. of Med. Chem. Vol. 10, n° 5, p.863-867 (1967)) describes as local anesthetics, various derivates, moreparticularly 2-ethoxycarbonyl-aceto-2′,6′-xylidide or its correspondingamide.

The Publication by McLaughlin et al. (J. of Neurochem. Vol. 70, p.2406-2415 (1998)) describes the effect of dopamine on the mitochbndrialinhibition compared to that of methylmalonate.

Patent Application WO97/16184 describes methods for treating ischemiacomprising the step of administrating an acyl-co-enzyme A cholesterol Oacyltransferase (ACAT) inhibitor such as dodecylN-(2,6-diisopropylphenyl)-2-phenyl-malomanic acid ester and aHMG-co-enzyme A reductase inhibitor.

AIMS OF THE INVENTION

The aim of the present invention is to provide new compounds designed toinduce a protective effect on protein complexes of the innermitochondrial membrane and designed to be used particularly forpreventing or treating partial or total ischemia, pathologies associatedwith ischemia or pathologies associated with mitochondrial deficiencies.

A particular aim of the present invention is to provide compounds withan improved activity and/or with no side effects of molecules in thestate of the art.

CHARACTERISTIC ELEMENTS OF THE INVENTION

The present invention relates particularly to new compounds of formulaI, the salts thereof and prodrugs of such compounds:

wherein:

R¹ is CH₂, NH or a ligand, preferably a carbon atom, forming togetherwith R³ and/or R⁴ an aromatic or non-aromatic ring having 5 or 6 carbonatoms (n=1 or 2), optionally comprising on each ring an heteroatom,preferably a nitrogen, oxygen or sulfur atom;

R³ and/or R⁴ represent:

an amine with the formula:

a group having the formula —Z—R⁶ wherein:

Z represents O or S,

R⁵ represents H or an alkyl group with 1 to 6 carbon atoms (preferably,R⁵ represents a methyl or ethyl group),

R⁶ represents an hydrophobic group, preferably selected amongst thegroup consisting of a tert-butyl group, an allyl group or a (aromatic ornon aromatic) ring having 5, 6 or 7 carbon atoms optionally comprisingone or more heteroatoms (preferably N, O or S) and wherein the carbonatoms are optionally substituted for by:

an alkyl group R⁷ having 1 to 6 carbon atoms (preferably, a methyl orethyl group),

a ketone group,

a hydroxyl group,

an ester group,

a halogen element (F, Cl, Br or I),

a trifluoromethyl (CF₃) group, or

a group having the formula:

wherein:

W represents CH or a nitrogen atom,

Y represents S or O, and

R⁸ represents an alkyl group having 1 to 6 carbon atoms (preferably amethyl or ethyl group),

with the additional condition that, if R³ forms together with R¹ saidabove-mentioned ring or if R³ is either an amine of formula:

R⁴ represents an alkyl group (preferably a propene) having 1 to 10(preferably 1 to 6) carbon atoms, either saturated or unsaturated,optionally comprising one or more heteroatoms (preferably N, S or O).Additionally, in said alkyl group, one or more carbons can optionally besubstituted for by an halogen element (fluorine, chlorine, bromine oriodine) or a trifluoromethyl group (CF3).

In the compounds having formula I, R³ and R⁴ preferably represent anamine having the formula:

a group having the formula —Z—R⁶ wherein R⁵ represents H or a methylgroup, Z represents O or S (preferably O) and R⁶ represents acyclopentene, a cyclohexane or a benzene group.

According to the invention, are excluded as compounds already describedin the state of the art, compounds having the formula I wherein R¹ isCH₂, R³ is a compound having the formula Z—R⁶ wherein Z represents O, R⁶represents an ethyl group, R³ is an amine having the formula NH—R^(6′)wherein R^(6′) represents a cyclic compound (optionally 2-substitutedwith an hydroxyl group) and compounds having formula I wherein R¹ isCH₂, R³ and R⁴ are amines having the formula NH—R⁶ wherein R⁶ representsa ring having 6 carbon atoms 2-substituted by an hydroxyl group.

Also excluded from the invention, as already described compounds, arethe compounds described in tables 1, 2, and A in the ApplicationWO87/05898, the dodecyl N-(2,6-diisopropylphenyl)-2-phenyl-malonamicacid ester and the compound having the formula I wherein R¹ is CH₂, R⁴is a group having the formula Z—R⁶ (Z represents oxygen and R⁶represents an alkyl group) and wherein R³ is an amine of formula II:

wherein X represents O, S, CH₂ or C═O and derivatives having formula:

wherein R¹ represents a C₁-C₆ alkyl group, A represents a saturated orunsaturated ring having 3 to 7 carbon atoms; each of R², R³ and R⁴represents, independently, a hydrogen or halogen atom or a group such asC₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ alkylthio, C₁-C₆ alkoxy-carbonyl,carboxyl, carboxamido, sulfonic acid, sulfonamido, acylamino,sulfonylamino, C₁-C₆ alkylsulfonyle, nitrile, nitro, acyloxy, phenyl ormethylene-dioxy groups and each of m and n represents an integer from 0to 4.

wherein:

R¹ represents an alkyl group, R² is hydrogen or an alkyl group.

wherein Z═CO methyl, COO methyl, or COO alkyl

Z═CO₂CH_(3,) CO₂C₂H₃ or CO₂H

wherein X═CON H and Y═H.

According to the invention, the aromatic or non aromatic ringsoptionally comprise one or more heteroatoms, preferably one or moreoxygen or sulfur atoms, and wherein the carbon atoms are substituted foreither by ketone, hydroxyl or ester groups or by an halogen element suchas described above.

The preferred compounds according to the invention are selected from thegroup comprised of dicyclopenten-2-yl malonate,2-propenyl-3-oxa-2-oxobicylo[3.1.0]hexane-1-carboxylate,ethylcyclopenten-2-yl malonate, methylcyclopenten-2-yl malonate,N,N′-diphenyl malonamide, ethyl-N-phenylamido ethanoate,ethyl-2,6-dimethyl-N-phenylamido ethanoate,ethyl-N-methyl-N-cyclohexylamido ethanoate and dicyclopentene-2-ylmalonate, optionally their salts or prodrugs thereof.

The present invention also relates to pharmaceutically acceptablenon-toxic acid addition salts of compounds according to the inventionhaving an amine. Examples of pharmaceutically acceptable acids mayinclude mineral acids, such as hydrochloric, hydrobromic, sulfuric,nitric, phosphoric acids, etc. and organic acids such as. acetic,citric, tartaric, benzoic, salicylic, maleic acids, etc.

The present invention also relates to the first application of compoundshaving formula I as a drug, as well as their salts and prodrugs of saidcompounds, except for the following known compounds: theN-(2,6-diisopropyl-phenyl)-2-phenyl-malonamic acid dodecyl ester and thecompounds having formula I wherein R¹ is CH₂, R⁴ is a group havingformula Z—R⁶ wherein Z represents oxygen and R⁶ represents an alkylgroup and wherein R³ is an amine of formula II:

wherein X represents O, S, CH₂ or C═O and the derivatives havingformula:

wherein R¹ represents a C₁-C₆ alkyl group, A represents a saturated oran unsaturated ring having 3 to 7 carbon atoms; each of R², R³ and R⁴represents, independently, a hydrogen or halogen atom, or a group suchas C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ alkylthio, C₁-C₆ alkoxycarbonyl,carboxyl, carboxamido, sulfonic acid, sulfonamido, acylamino,sulfonylamino, C₁-C₆ alkylsulfonyl, nitrile, nitro, acyloxy, phenyl ormethylene-dioxy groups and each of m and n represents an integer from 0to 4.

wherein R¹ represents an alkyl group, R² hydrogen or an alkyl group.

According to the invention, it is meant by “prodrugs” functionalderivatives of formula I compounds that may be converted, preferably invivo, in the patient depending on the required formula I form. Suchprodrugs of these compounds can be obtained using methods well-known tothe man of the art such as those described in the document entitled:“Design and Prodrugs”, Ed. H. Bundgaard, Elsevier (1985). The compoundsaccording to the invention relate to two enantiomeric forms, the variousseparate isomers or a mixture thereof.

The new compounds according to the invention are advantageously used inorder to obtain a protective effect on the protein complexes of theinner mitochondrial membrane of a mammalian's cells, preferably a humanbeing. The compounds according to the present invention are moreparticularly applied to prevent and/or treat partial or total ischemia,pathologies associated with ischemia or pathologies associated withmitochondrial deficiencies.

It is meant by “(partial or total) ischemia, pathologies associated withischemia and pathologies associated with mitochondrial deficiencies”,diseases, preferably vascular ones, selected amongst the groupconsisting of myocardial infarction, cerebral ischemia, chronic veinousinsufficiency, arteriopathies, i.e. lesions due to atherosclerosisaffecting patients' arteries, more particularly those of the lowerlimbs, the Raynaud's phenomenon linked to vasospasms, leading to anartery vasoconstriction, ulcers, gangrene, alteration of the capillarypermeability, capillary fragility, cicatrizations, skin alterations,retinal defects from ischemic origin, decrease of the auditive acuityfrom ischemic origin, troubles associated with stays in high altituderegions, angina pectoris caused by short periods of coronaryobstruction, pulmonary hypertension, hepatic ischemia, Parkinsondisease, myopathies and syndroms associated with vascular problems, suchas diabetes, where a hypertension and an alteration of the blood flowappear in the lower limbs. Such pathologies and diseases linked toischemia are well-known to the clinicians and practitioners who are ableto adapt the use of the pharmaceutical composition for treating and/orpreventing symptoms and dysfunctions of the human or animal bodyassociated with the above-mentioned diseases and/or for preventing orreducing the possibility of being affected by them.

The Applicants have unexpectedly found out that such various syndroms ordiseases can be treated using the compounds of the invention, theiroptional salts and prodrugs thereof, and that such compounds actaccording to the same biochemical action mode.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising an appropriate pharmaceutical carrier orexcipient and a sufficient amount of one or more compounds according tothe invention, i.e. an amount sufficient to at least improve or preventthe above-mentioned symptoms in a mammalian, more particularly a humanbeing. Such a sufficient amount may vary depending on some factors suchas the condition of the animal to be treated, the administration route,the side-effect severity, the compound stability in the circulatingserum or blood, etc. Preferably, the compound sufficient amount to beused in the pharmaceutical composition of the invention is in the rangebetween 0.1 and 200 mg/patient, more preferably between 1 and 50mg/patient, most preferably between 20 and 30 mg/patient, this amountbeing optionally able to be adapted particularly depending on thenecessary administration doses and the patient's weight, as this may beextrapolated from in vivo application examples, as disclosedhereinafter.

The pharmaceutical composition comprises an appropriate pharmaceuticalcarrier that can vary according to the administration mode and can bepossibly combined with an adjuvant, so as to improve the therapeuticproperties of the compound according to the invention or to reduce itspossible side effects. Such appropriate pharmaceutical carriers oradjuvants are well-known to the man of the art and can be preparedfollowing the procedures generally applied by chemists and may compriseany non toxic pharmaceutical carrier, either solid (including in powderform), liquid (solutions, suspensions, emulsions, etc.) or gaseous. Theactive pharmaceutical compound percentage (generally in the rangebetween 5% and 70% in weight) may vary depending on the administrationfrequency and the possible side-effects on animal, including on humanbeing.

In order to prepare such pharmaceutical compositions in the form oftablets, granules, capsules or tablets, suspensions, etc., it ispossible to incorporate elements such as corn starch, lactose, sucrose,sorbitol, talcum, stearic acid, magnesium stearate, gums or diluentscomprising a variable percentage of water or a solvent such as a syrup,oil or water suspensions, perfumed emulsions, etc. Dispersible agentsused in aqueous compositions may comprise gums, alginates, dextrans,carboxymethyl cellulose derivatives, etc.

The present invention also relates to a therapeutic or preventivetreatment method for ischema or pathologies associated with ischemia asabove-mentioned, which includes administering a sufficient amount offormula I compound or of the pharmaceutical composition of the inventionto said patient, preferably a human being, so as to prevent, reduce oreliminate the symptoms of ischemia, of pathologies associated withischemia or associated with mitochondrial deficiencies, or apoptosis,with the additional condition that the compound is neither the dodecylN-(2,6-diisopropyl-phenyl-)-2-phenyl-malonamic acid ester nor a formulaI compound wherein R¹ is CH₂, R⁴ is a group having the formula —Z—R⁶wherein Z represents oxygen and R⁶ represents an alkyl group and whereinR³ is an amine having formula II:

wherein X represents O, S, CH_(2,) C═O.

A last aspect of the present invention relates to using such compoundsor the pharmaceutical composition of the invention for preparing a drugfor treating or preventing ischemia, pathologies associated withischemia or associated with mitochondrial deficiencies, such asabove-mentioned, or apoptosis, with the additional condition asmentioned in the preceding paragraph.

The present invention also relates to a method for preparing compoundsof the invention having formula I, comprising the following steps:

esterification of an intermediate compound R³—H by a group of thefollowing formula:

in the presence of dicyclohexyl carbodiimide (DCC),

optionally followed by one or more cyclizing reactions in the presenceof an oxidizing agent such as manganese acetate Mn(III) or monohydratecopper acetate Cu(II), and optionally sodium acetate.

The definitions of the above-mentioned moieties are those given informula I for the compounds of the invention.

The various aspects of the present invention are described more indetails in the non limitative examples presented hereinafter.

EXAMPLES Example 1 Dicyclopenten-2-yl malonate Synthesis

2-Cyclopentenol Synthesis

Such synthesis has been conducted according to the method described byA. L. Gemal and J. L. Luche (J. Am. Chem. Soc. 103, p. 5454 (1981)).

To a 2-cyclopentenone solution (5.00 g; 60.90 mmols) in (commercial,99%) methanol is added heptahydrate cerium chloride (CeCl₃.7H₂O) (22.69g; 60.90 mmols) and the reaction mixture is stirred at room temperaturefor 40 minutes for homogeneization. The mixture is then cooled down to0° C. and sodium borohydride (NaBH₄) (2.30 g; 60.90 mmols) is slowlyadded. After complete addition, the mixture is stirred at 0° C. for 3hours. A hydrochloric acid diluted solution is then added in order toadjust the pH to 7. The phases are separated and the aqueous phase isextracted several times with ether (5×50 ml). The combined organicphases are dried on anhydrous MgSO₄ and concentrated in order to givethe 2-cyclopentenol (3.81 g; 74%).

Dicyclopenten-2-yl malonate Synthesis

To a 2-cyclopentenone solution (1.00 g; 11.89 mmols) in 50 mldichloromethane are successively added malonic acid (1.12 g; 10.81mmols) and 4-dimethylamino pyridine (DMAP) (0.13 g; 1.08 mmols). Thereaction mixture is stirred under inert atmosphere and cooled down to 0°C. At this temperature, dicyclohexyl carbodiimide (DCC) (2.34 g; 11.35mmols) is added in one single portion. A milky white mixture isobtained, which is stirred at 0° C. for 1 hour, followed by roomtemperature until complete reaction (reaction evolution monitoring byTLC, eluent pentane/ethyl acetate: 8/2). The precipitate is filtered outon celite and washed with dichloromethane. The filtrate is washed with aNaHCO₃ (10 ml) saturated aqueous solution. The phases are separated andthe aqueous phase is extracted with dichloromethane (3×10 ml). Thecombined organic phases are successively washed with a NaCl saturatedaqueous solution and water, then dried on anhydrous MgSO₄ andconcentrated. The precipitate is again filtered out through filtrationon celite and washed with pentane. The purification of the reaction rawproduct is performed by silica column chromatography (elutionpentane/ethyl acetate: 8/2) to give MRC2P119 (2.40 g; 94%) in the formof a colourless oil.

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ: 1.81-2.55 (m, 8H), 3.32 (s, 2H), 5.76-5.84 (m,4H), 6.12 (m, 2H); ¹³C NMR (CDCl₃, 175 MHz)δ: 29.05, 30.53, 41.36,80.97, 128.33, 137.44, 165.92; IR (film): 3,450, 3,062, 2,947, 2,858,1,727, 1,618, 1,454, 1,411, 1,348, 1,319, 1,270, 1,151, 1,029, 973, 917,878, 783, 735 cm⁻¹; PM: 236.27; MS m/z 171 (M⁺−65); calculatedelementary analysis C (66.09%), H (6.82%); found C (66.09%), H (6.90%).

Example 2 2-propenyl-3-Oxa-2-Oxobicyclo[3.1.0]-hexane-1-carboxylate

Diallyl Malonate Synthesis

To a malonic acid solution (5.00 g; 48.05 mmols) in 330 mldichloromethane are successively added allyl alcohol (10.51 g; 124.93mmols) and DMAP (1.17 g; 9.61 mmols). The reaction mixture is stirredunder inert atmosphere and cooled down to 0° C. At this temperature, DCC(20.82 g; 100.90 mmols) is added in one single portion. A milky whitemixture is obtained, which is stirred at 0° C. for 1 hour, followed byroom temperature until complete reaction (reaction evolution monitoringby TLC, eluent pentane/ethyl acetate: 9/1). The precipitate is filteredout on celite and washed with dichloromethane. The filtrate is washedwith a NaHCO₃ (20 ml) saturated aqueous solution. The phases areseparated and the aqueous phase is extracted with dichloromethane (3×20ml). The combined organic phases are successively washed with a NaClsaturated aqueous solution and water, then dried on anhydrous MgSO₄ andconcentrated. The precipitate is again filtrated on celite and washedwith pentane. The purification is performed by silica columnchromatography (elution pentane/ethyl acetate: 9/1) to give the compoundof the invention (MRC2PSS) (8.09 g; 92%) in the form of a colourlessoil.

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ: 3.44 (s, 2H), 4.64 (d, J=5.9 Hz, 4H), 5,24 (d,J=10.3 Hz, 2H), 5.34 (d, J=17.1 Hz, 2H), 5.91 (ddt, J=17.1-10.3-5.9 Hz,2H); ¹³C NMR (CDCl₃, 175 MHz)δ: 40.99, 65.57, 118.20, 131.26, 165.68; IR(film): 3,653, 3,089, 2,989, 2,951, 1,739, 1,650, 1,450, 1,414, 1,368,1,330, 1,274, 1,150, 995, 935, 631 cm⁻¹; PM: 184.19; MS m/z 184 (M³⁰);calculated elementary analysis C (58.69%), H (6.57%), found C (58.66), H(6.57%).

2-Propenyl-3-oxa-2-oxobicyclo[3.1.0]hexane-1-carboxylate Synthesis

To a diallyl malonate solution (MRC2P55) (2.00 g; 10.86 mmols) in 55 mlicy acetic acid are successively added dihydrate. manganese(III) acetate(Mn(OAc)₃.2H₂O) (11.64 g; 43.43 mmols), monohydrate copper(II) acetate(Cu(OAc)₂.H₂O) (4.34 g; 21.72 mmols) and sodium acetate (AcONa) (1.78 g;21.72 mmoles). The resulting brown solution is heated at 80° C. for 20hours (this corresponds to the brown coloration disappearance and theappearance of a turquoise blue coloration). The reaction evolution ismonitored by TLC (eluent pentane/ethyl acetate: 9/1). The precipitate isfiltered out on celite and washed with dichloromethane. The filtrate iswashed with water (30 ml). The aqueous phase is extracted withdichloromethane (2×20 ml). The combined organic phases are successivelywashed with water (2×20 ml) and a NaHCO₃ saturated aqueous solution(2×20 ml), then dried on anhydrous MgSO₄ and concentrated. The reactionraw product is purified by silica column chromatography (elutionpentane/ethyl acetate: 1/1) to give the compound of the invention(MCR2PS7) (0.49 g; 25%) in the form of a colourless oil.

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ: 1.41 (dd, J=5.4-4.9 Hz, 1H), 2.10 (dd,J=8.0-4.6 Hz, 1H), 2.79 (m, 1H), 4.20 (d, J=9.3 Hz, 1H), 4.38 (dd,J=9.3-4.9 Hz, 1H), 4.68 (d, J=5.4 Hz, 2H), 5.28 (dd, J=10.2-1.5 Hz, 1H),5.39 (dd, J=17.1-1.5 Hz, 1H), 5.94 (ddt, J=17.1-10.2-5.4 Hz, 1H); ¹³CNMR (CDCl₃, 175 MHz)δ: 20.56, 27.82, 29.09, 65.97, 66.85, 118.60,131.10, 166.14, 170.20; IR (film) 3,653, 3,094, 2,989, 1,777, 1,725,1,650, 1,447, 1,393, 1,314, 1,271, 1,183, 1,115, 1,090, 1,047, 998, 939,847, 793, 765, 703, 632 cm⁻¹; PM: 182.18; MS m/z 183 (M⁺); calculatedelementary analysis C (59.34%), H (5.53%), found C (59.28%), H (5.68%).Example 3

Ethyl Cyclopenten-2-yl malonate Synthesis

Ethyl Malonic Acid Synthesis

Step 1

In a 250 ml three neck balloon provided with an addition bulb and arefrigerant, diethyl malonate is solubilized (20.00 g; 124.87 mmols) in(commercially available) ethanol. The resulting colourless mixture isstirred at room temperature and a KOH solution (7.00 g; 124.76 mmols) inethanol (80 ml) is slowly added (addition time: 1 hr). After completeaddition, the resulting mixture is stirred at room temperature overnight(ethyl potassium malonate precipitation). The salt precipitation isadvantageously accelerated by cooling the reaction balloon in an icebath. The potassium salt is recovered through filtration, washed withsmall ether quantities, then dried under reduced pressure (17.77 g; 84%)

Step 2

In a 250 ml three neck balloon provided with a magnetic stirrer, anaddition bulb and a thermometer, the ethyl potassium malonate (17.77 g;104.49 mmols) is solubilized in 18 ml water. The reaction mixture iscooled by means of an ice bath and 8, 2 ml concentrated hydrochloricacid are slowly added. The reaction mixture is filtered and the KClprecipitate is washed with ether (3×50 ml). The filtrate is allowed tosettle and the aqueous phase is extracted with ether (3×50 ml). Thecombined ether phases are dried on anhydrous MgSO₄, filtered andconcentrated to give the ethyl malonic acid (13.45 g; 82% global yieldfor both steps) in the form of a colourless oil.

Ethyl Cyclopenten-2-yl malonate Synthesis

To a 2-cyclopentenol solution (0.70 g; 8.32 mmols) in 30 mldichloromethane are successively added ethyl malonic acid (0.85 g; 6,40mmols.) and DMAP (0.08g; 0.64 mmol). The reaction mixture is stirredunder inert atmosphere and cooled down to 0° C. At this temperature, DCC(1.39 g; 6.72 mmols) is added in one single portion. A milky whitemixture is obtained, which is stirred at 0° C. for 1 hour, followed byroom temperature until complete reaction (reaction evolution monitoringby TLC, eluent pentane/ethyl acetate: 8/2). The precipitate is filteredout on celite and washed with dichloromethane. The filtrate is washedwith a NaHCO₃ (10 ml) saturated aqueous solution. The phases areseparated and the aqueous phase is extracted with dichloromethane (3×10ml). The combined organic phases are successively washed with a NaClsaturated aqueous solution and water, then dried on anhydrous MgSO₄ andconcentrated. The precipitate is again filtered out on celite and washedwith pentane. The purification of the reaction raw product is performedby silica column chromatography (elution pentane/ethyl acetate: 8/2) togive the compound of the invention (MRC2P45) (1.39 g; 84%) in the formof a colourless oil.

Product Characterization ¹H NMR (CDCl₃, 400 MHz)δ: 1.28 (t, J=7.3 Hz,3H), 1.83 (m, 1H), 2.31 (m, 2H), 2.50 (m, 1H), 3.34 (s, 1H), 4.20 (q,J=7.3 Hz, 2H), 5.76 (m, 1H), 5.83 (m, 1H), 6.13 (m, 1H); ¹³C NMR (CDCl₃,175 MHz)δ: 13.41, 29.00, 30.47, 41.10, 60.64, 81.07, 128.46, 165.87,165.94; IR (film): 3,451, 2,983, 1,750, 1,731, 1,459, 1,412, 1,370,1,346, 1,323, 1,271, 1,189, 1,151, 1,032, 971, 917, 879, 736 cm⁻¹; PM:198.22; MS m/z 170 (M⁺−28).

Example 4 Methyl Cyclopenten-2-yl malonate Synthesis

Methyl Malonic Acid Synthesis

Step 1

In a 100 ml three neck balloon provided with an addition bulb and arefrigerant, dimethyl malonate is solubilized (10.00 g; 75.69 mmols) in40 ml (commercially available) methanol. The resulting colourlessmixture is stirred at room temperature and a KOH solution (4.25 g; 75.69mmols) in methanol (40 ml) is slowly added (addition time: 1 hr). Aftercomplete addition, the resulting mixture is stirred at room temperatureovernight (methyl potassium malonate precipitation). The saltprecipitation can be accelerated by cooling the reaction balloon in anice bath. The potassium salt is recovered through filtration, washedwith small ether quantities, then dried under reduced pressure (10.27 g;87%).

Step 2

In a 50 ml three neck balloon provided with a magnetic stirrer, anaddition bulb and a thermometer, methyl potassium malonate (10.27 g;65.76 mmols) is solubilized in 10 ml water. The reaction mixture iscooled by means of an ice bath and 5.5 ml concentrated hydrochloric acidare slowly added. The reaction mixture is filtered and the KClprecipitate is washed with ether (3×10 ml). The filtrate is allowed tosettle and the aqueous phase is extracted with ether (3×10 ml). Thecombined ether phases are dried on anhydrous MgSO₄, filtered andconcentrated to give the methyl malonic acid (7.42 g; 96%) in the formof a colourless oil.

Methyl cyclopenten-2-yl malonate Synthesis

To a 2-cyclopentenol solution (1.00 g; 11.89 mmols) in 35 mldichloromethane are successively added methyl malonic acid (1.08 g; 9.15mmols) and DMAP (0.11 g; 0.91 mmols). The reaction mixture is stirredunder inert atmosphere and cooled down to 0° C. At this temperature, DCC(1.98 g; 9.60 mmols) is added in one single portion. A milky whitemixture is obtained, which is stirred at 0° C. for 1 hour, followed byroom temperature until complete reaction (reaction evolution monitoringby TLC, eluant pentane/ethyl acetate: 8/2). The precipitate is filteredout on celite and washed with dichloromethane. The filtrate is washedwith a NaHCO₃ (10 ml) saturated aqueous solution. The phases areseparated and the aqueous phase is extracted with dichloromethane (3×10ml). The combined organic phases are successively washed using a NaClsaturated aqueous solution and water, then dried on anhydrous MgSO₄ andconcentrated. The precipitate is again filtered out on celite and washedwith pentane. The purification of the reaction raw product is performedthrough silica column chromatography (elution pentane/ethyl acetate:8/2) to give the compound of the invention (MCR2P85) (1.40 g; 83%) inthe form of a colourless oil.

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ: 1.85 (m, 1H), 2.29 (m, 2H), 2.51 (m, 1H), 3.36(S, 2H), 3.75 (s, 3H), 5.76 (m, 1H), 5.83 (m, 1H), 6.13 (m, 1H); ¹³C NMR(CDCl₃, 175 MHz)δ: 29.27, 30.73, 41.16, 52.04, 81.43, 128.44, 137.83,166.10, 166.74; IR (film): 2,955, 2,858, 1,736 1,438, 1,412, 1,348,1,273, 1,152, 1,028, 968, 917, 878, 737 cm⁻¹; PM: 184.19; MS m/z 156(M⁺−28); calculated elementary analysis C (58.69%), H (6.57%), found C(58.60%), H (6.59%).

Example 5 N-N′-diphenyl malonamide Synthesis

To a N-phenylamine solution (0.75 ml; 8.22 mmols) in 25 mldichloromethane is added triethylamine (0.72 ml; 5.14 mmols). Theresulting colourless mixture is cooled down to 0° C., using an ice bathand malonyl dichloride (0.5 ml; 5.14 mmols) is slowly added to through asyringe. A vivid red/orange solution accompanied with a precipitate isobtained. The reaction mixture is stirred at 0° C. for 2 hours (reactionevolution monitoring by TLC, eluent pentane/ethyl acetate: 1/9),followed by the addition of approximately 4 ml of a 10 HCl solution. Thephases are separated and the organic phase is successively washed withwater (10 ml) and with a NaCl (10 ml) saturated aqueous solution. Afterbeing dried on anhydrous MgSO₄, filtered and concentrated, anorange-coloured solid is isolated. The purification is performed byrecrystallization in a pentane/ethyl acetate blend in order to give thecompound of the invention (MCR2P219) (0.33 g; 32%) in the form of anecru-coloured powder.

Product Characterization

¹H NMR (DMSO-d₆, 400 MHz)δ: 3.47 (s, 2H), 7.06 (m, 2×1H), 7.31 (m,2×2H), 7.60 (m, 2×2H), 10.17 (s, 2H); ¹³C NMR (DMSO-d₆, 175 MHz)δ:37.20, 110.30, 114.60, 120.00, 130.20, 156.60; IR (KBr tablets): 3,274,3,152, 3,066, 1,669, 1,650, 1,599, 1,560, 1,538, 1,500, 1,444, 1,416,1,358, 1,309, 1,294, 1,251, 1,193, 1,162, 979, 905, 857, 752, 693, 618cm⁻¹; P.F.=220° C.; PM 254.29.

Example 6 Ethyl N-phenylamido Ethanoate Synthesis

Ethyl Chloroformyl Ethanoate Synthesis

In a 50 ml three neck balloon provided with an addition bulb, athermometer and a refrigerant, ethyl malonic acid is introduced (1.00 g;7.57 mmols) in 8 ml dry-dichloromethane. The balloon is cooled down to0° C. using an ice bath. At this temperature, 1.5 thionyl chlorideequivalents (0.83 ml;. 11.35 mmols) diluted in 5 ml dichloromethane areslowly added. After complete addition, the reaction mixture is stirredat 0° C. for 10 minutes, subsequently brought to reflux for 2 hours.After concentration, the ethyl chloroformyl ethanoate (0.99 g; 87%) isisolated and used with no additional purification in the next step.

Product Characterization ¹H RMN (CDCl₃, 400 MHz)δ: 1.31 (t, J=7.3 Hz,3H), 3.87 (s, 2H), 4.26 (q, J=7.3 Hz, 2H); IR (film): 2,989, 1,802,1,742, 1,577, 1,468, 1,401, 1,372, 1,323, 1,260, 1,164, 1,035, 982, 940,859, 652, 612 cm⁻¹; PM: 150.56.

Ethyl N-phenylamido Ethanoate Synthesis

To a N-phenylamine solution (0.25 g; 0.24 ml, 2.66 mmols) in 15 ml drydichloromethane, are added 1.25 triethylamine equivalents (0.46 ml; 3.32mmoles). The resulting solution is cooled down to 0° C. using an icebath. At this temperature, 1.25 ethyl chloroformyl ethanoate equivalents(0.43 ml; 3.32 mmols) are slowly added through a syringe. A vivid orangesolution is obtained, which is stirred at 0° C. overnight (reactionevolution monitoring by TLC) (eluent pentane/ethyl acetate: 1/9),followed by the addition of approximately 4 ml of a 10% HCl solution.Both phases are separated and the organic phase is washed with water.The combined aqueous phases are extracted with dichloromethane. Theorganic phases are washed with a NaCl saturated aqueous solution, driedon anhydrous MgSO₄, filtered and concentrated. The purification isperformed through silica column chromatography (eluent pentane/ethylacetate: 1/9) to give quantitatively the compound of the invention(MCR2P231) (0.55 g; 100%).

Product Characterization ¹H NMR (CDCl₃, 400 MHz)δ: 1.32 (t, J=7.3 Hz,3H), 3.47 (s, 2H), 4.26 (q, J=7.3 Hz, 2H), 7.12 (t, J=7.3 Hz, 1H), 7.33(t, J=7.8 Hz, 2H), 7.55 (d, J=7.8 Hz, 2H); ¹³C NMR (CDCl₃, 175 MHz)δ:13.67, 42.26, 61.38, 119.98, 124.25, 128.55, 137.37, 163.77, 168.70; IR(film): 3,313, 3,143, 2,985, 1,738, 1,668, 1,603, 1,549, 1,500, 1,445,1,372, 1,342, 1,244, 1,190, 1,156, 1,033, 906, 844, 757, 695 cm⁻¹; PM:207,23; MS m/z 207 (M⁺).

Example 7 Ethyl 2,6-dimethyl-N-phenylamido Ethanoate Synthesis

To a 2,6-dimethyl-N-phenylamine solution (1.31 ml; 10.63 mmols) in 60 mldry dichloromethane is added triethylamine (1.85 ml; 13.28 mmols). Theresulting colourless mixture is cooled down to 0° C. through an ice bathand ethyl chloroformyl ethanoate (1.3 ml; 10.15 mmols) is then slowlyadded through a syringe. A vivid red/orange solution is obtained, whichis maintained under stirring at 0° C. overnight (reaction evolutionmonitoring by TLC, eluent pentane/ethyl acetate: 1/9), followed by theaddition of approximately 4 ml of a 10% HCl solution. Both phases areseparated and the organic phase is washed with water (3×10 ml). Thecombined aqueous phases are extracted with dichloromethane. The organicphase is washed with a NaCl saturated aqueous solution, then dried onanhydrous MgSO₄. After being filtered and concentrated, the raw esteramide is obtained in the form of an orange-coloured solid. Thepurification is performed by recrystallization in a pentane/ethylacetate blend in order to give thee compound of the invention (MCR2P255)(1.67 g; 67%) in the form of an ecru-coloured powder.

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ: 1.35 (t, J=7.3 Hz, 3H), 2.20 (s, 6H), 3.50 (s,2H), 4.25 (q, J=7.3 Hz, 2H), 7.10 (m, 3H), 8.55 (broadened s, 1H); ¹³CNMR (CDCl₃, 175 MHz)δ: 13.98, 18.29, 41.20, 61.67, 127.23, 128.06,133.50, 135.10, 163.38, 169.66; IR (KBr tablet): 3,226, 3,031, 1,731,1,666, 1,642, 1,595, 1,536, 1,477, 1,411, 1,397, 1,370, 1,351, 1,264,1,218, 1,204, 1,166, 1,030, 986, 880, 761, 723, 635 cm⁻¹; P.F.=103° C.;PM: 235.28; MS m/z 235 (M⁺)

Example 8 Ethyl N-methyl -N-cyclohexamido Ethanoate Synthesis

To a N-methyl N-cyclohexylamine solution (0.41 ml; 3.12 mmols) in 20 mldry dichloromethane is added triethylamine (0.54 ml; 3.91 mmols). Theresulting colourless mixture is cooled down to 0° C. through an ice bathand then ethyl chloroformyl ethanoate (0.5 ml; 3.91 mmols) is slowlyadded through a syringe. A vivid red/orange solution is obtained whichis maintained under stirring at 0° C. for 4 hours (reaction evolutionmonitoring by TLC, eluent pentane/ethyl acetate: 1/9), followed by theaddition of approximately 4 ml of a 10% HCl solution. Both phases areseparated and the organic phase is washed with water (3×10 ml). Thecombined aqueous phases are extracted with dichloromethane. The organicphase is washed with a NaCl saturated aqueous solution, followed bydrying on anhydrous MgSO₄. After being filtered and concentrated, theraw ester amide is obtained. The purification is performed throughsilica column chromatography (eluent pentane/ethyl acetate: 1/9) to givethe compound of the invention (MCR2P237) (0.71 g; 100%) in the form ofan orange liquid.

Characterization of the Resulting Product

¹H NMR (CDCl₃, 400 MHz)δ: 1.27-1.67 (m, 14H), 2.84 (s, 3H) 3.44 (s, 1H),3.47 (s, 1H), 4.21 (q, J=7.3 Hz, 2H), 4.40 (m, enol form contribution);¹³C NMR (CDCl₃, 175 MHz)δ: 14.52, 24.99, 29.05, 30.14, 41.44, 52.09,60.48, 65.10, 165.13, 167.06; IR (film) 2,933, 2,858, 1,741, 1,645,1,477, 1,450, 1,408, 1,370, 1,326, 1,254, 1,164, 1,098, 1,035, 947, 895,847, 788, 674 cm⁻¹; PM: 227.30.

Example 9 S,S dicyclopenten-2-yl Malonate Enantiomer Synthesis

2-Bromo-2-cyclopentenone Synthesis

In a 250 ml three neck balloon provided with an addition bulb and arefrigerant, 2-cyclopentenone is solubilized (7.24 g; 88.2 mmole) in 60ml CCl₄. The solution is cooled down to 0° C. and a Br₂ solution (15.51g; 97 mmols) in the CCl₄ is added dropwise. After complete addition, aN-Et₃ solution (13.39 g; 132 mmols) in the CCl₄ (60 ml) is added at thesame temperature dropwise. The resulting mixture is stirred at roomtemperature for 2 hours, then filtered. The filtrate is successivelywashed with HCl 1M, 10% NaHCO₃ and H₂O. The organic phase is dried onanhydrous MgSO₄, filtered and concentrated. The thereby obtained solidis recrystallized (cyclohexane/Et₂O) to give the2-bromo-2-cyclopentenone (7.5 g; 53%) in the form of a white solid.

(±)-2-bromo-2-cyclopentenol Synthesis

To a 2-bromo-2-cyclopentenone solution (8 g; 49.7 mmols) in methanol(150 ml) is added heptahydrate cerium chloride (CeCl₃.7H₂O) (20.36 g;54.7 mmols) and the reaction mixture is stirred at room temperature for40 minutes. The mixture is then cooled down to 0° C. and sodiumborohydride (NaBH₄) (2.07 g; 54.7 mmoles) is slowly added. Aftercomplete addition, the mixture is stirred at 0° C. for 2 hours. A NH₄Claqueous solution is then added. The phases are separated and the aqueousphase is extracted several times with ether (5×50 ml). The combinedorganic phases are dried on anhydrous MgSO₄ and concentrated. The rawproduct thereby obtained is purified with silica chromatography (elutionpentane/ethyl acetate: 9/1) to give the (±)-2-bromo-2-cyclopentenol (6.0g; 74%) in the form of a yellow oil.

Product Characterization

¹H NMR (CDCl₃, 400 MHz) 1.85-2.04 (m, 2H), 2.20-2.60 (m, 3H), 4.71 (b,1H), 6.05-6.08 (m, 1H)

(S)-2-bromo-2-cyclopentenol Synthesis

To a (±)-2-bromo-2-cyclopentenol solution (6.00 g; 36.8 mmols) in amixture of isopropenylacetate (24 ml) and cyclohexane (96 ml) is addedthe Novozym Lipase enzyme 435 (1,5 g). The mixture is stirred for 1 hourat room temperature, then filtered. The filtrate is evaporated and thethereby obtained residue is purified through silica chromatography(elution cyclohexane/AcOEt: 9/1) to give the (S)-2-bromo-2-cyclopentenol(1.5 g; 50%) in the form of a white solid.

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ: 1.85-2.04 (m, 2H), 2.20-2.60 (m, 3H), 4.71,(b,1H), 6.05-6.08 (m, 1H). [α]²²D=−47.5° (c=1.0, CHCl₃)

(S)-2-cyclopentenol Synthesis

In a 50 ml three neck balloon provided with an addition bulb, athermometer and a refrigerant, tBuLi (solution in pentane 1.7M; 23 ml;39.1 mmols) and Et₂O (10 ml) are introduced. The mixture is then cooleddown to −78° C. and (S)-2-bromo-2-cyclopentenol (1.90 g; 11.7 mmols) insolution in Et₂O (15 ml) is slowly added. After complete addition, themixture is allowed to heat at 0° C. and is stirred at this temperaturefor 2 hours. A diluted NH₄Cl solution in water is then slowly added. Theorganic phase is separated and the aqueous phase extracted several timesusing Et₂O. The combined organic phases are dried on anhydrous MgSO₄,and concentrated to give the (S)-2-cyclopentenol in the form of oil (600mg; 61%).

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ: 1.65-1.73 (m, 2H), 2.22-2.32 (m, 2H),2.48-2.56 (m, 1H), 4.87 (b, 1H), 5.81-5.89 (m, 1H), 5.90-6.10 (m, 1H)[α]²²D −119.2° (c=1,0, CHCl₃)

Di-(S)-2-cyclopenten-2-yl Malonate Synthesis

The synthesis of di-(S)-2-cyclopenten-2-yl malonate is performed usingthe same protocol as described in example 1.

Product Characterization

¹H NMR (CDCl₃, 400 MHz)δ:: 1.80-1.92 (m, 2H), 2.20-2.40 (m, 4H),2.45-2.60 (m, 2H), 3.34 (s, 2H), 5.75-7.79 (m, 2H), 5.83-5.86 (m, 2H),6.10-6.17 (m, 2H) [α]²²D=212.8° (c=0,97, CHCl₃)

Example 10 In vitro Tests

Effect of the Products at the Mitochondrial Respiration Level (Effect atthe Level of Stages III and IV and at the RCR Level

The compounds of the invention are characterized by the inhibition ofthe reduction of the ATP content, the activation of the A2 phospholipaseand the neutrophilic polymorphonuclear adherence (PMN) on the culturedendothelial cells of the human umbilical vein when they are incubated inhypoxic conditions.

The inhibition by the compounds of the invention of the activationcascade of endothelial cells induced by hypoxia and the ATP contentpreservation in the endothelial cells under hypoxic conditions areobtained by the compounds of the invention that hold the mitochondrialrespiratory activity. This is confirmed by measuring the respiratoryactivity expressed by the respiratory control (RCR) of rat livermitochondria treated per os. The mitochondria are isolated according tothe method described by Nowicki et al., J. of Cerebral Blood Flow andMetabolism 2, p. 33-40 (1982). The mitochondrial respiration is measuredby an oxygen electrode connected to a recorder. The RCR represents therespiratory control, and represents the ratio of the oxygen consumptionin the presence of an endogenous substrate (glutamate/malate) to theconsumption after ADP phosphorylation in ATP. This technique has beendescribed by Chance & William (Nature 175, p. 1120-1121 (1955)).

Several compounds of the invention have been tested at variousconcentrations on the respiratory activity of purified mitochondriaobtained from rat livers. The mitochondria have been preincubated in thepresence of the compounds so as to measure the slope in respirationstages III and IV, as described in WO98/51291. The RCR has beensubsequently calculated.

The results hereafter show that the products of example 1 and example 2have an action on the RCR with a maximum effect at 10⁻⁷ mol/l for theexample 2 product. This RCR increase is mainly due to a decrease effectof respiration stage IV.

The compound of example 1 significatively increases the mitochondriaRCR, namely because this molecule strongly decreases respiration stageIV. The effect is optimal at 10⁻⁶ mol/l and decreases with theconcentration to become negligible at 10⁻⁹ mol/l.

The table below summarizes the effect of other compounds of theinvention with the optimum concentrations at which the effects have beenobserved.

TABLE I Compound RCR increase (*) Concentration Ethyl cyclopenten-2-yl10% 10⁻⁵ M malonate Methyl cyclopenten-2-yl 15% 10⁻⁶ M malonatedi-(S)-cyclopenten-2-yl 18% 10⁻⁶ M malonate N,N′-diphenylmalonamide  5%10⁻⁷ M Ethyl N-phenlyamido 6.6% 10⁻⁶ M ethanoate Ethyl 2,6-dimethyl-N-13% 10⁻⁶ M phenylamido ethanoate Ethyl N-methyl-N-cyclo- 15% 10⁻⁷ Mhexamido ethanoate (*) In % relative to the control brought to 100%

The results show that the compounds of the invention are able to protectreproducibly the decrease in ATP content induced by hypoxia in theendothelial cells. The 92.5% average maximum protection is observed at10⁻⁴ mol/l concentration for the example 2 compound. This protectiondecreases with this compound concentration to become negligible at 10⁻⁶mol/l. The results also show that the compound from example 1 is able toprotect reproducibly the decrease in ATP content induced by hypoxia inthe endothelial cells. The 92.1% average maximum protection is observedat 10⁻⁴ mol/l to 10⁻⁵ mol/l concentration. This protection decreaseswith this compound concentration to become negligible at 10⁻⁸ mol/l.

This protection is reproducible as long as the same curve evolutions arealways found for all the experiments, and even if there is a variabilitybetween experiments at the level of the hypoxia effect on the ATPcontent. This variability is inherent to the experimental model. Indeed,for each experiment, a different culture is used from differentumbilical cord isolated cells and there is a behaviour variability ofthe cells from one cord to another.

Example 11 Stability in Human Plasma

The compounds of examples 1 and 2 have been submitted to stability testsin order to evaluate their behaviour when in contact with plasma after a3 and 24 hour incubation at a temperature of 37° C. These tests havebeen performed using GC following the evolution of chromatograms of thedifferent esters compared to an internal standard as a function of time.

The injection of GC/MF pure products has been performed in the followingconditions:

injection temperature: 250° C.

initial temperature: 40° C.

final temperature: 250° C.

temperature gradient: 10° C./mn

injection method: split

retention time: compound of example 1: 13.39

compound of example 2: 11.23.

The different compounds have been prepared as follows: 10 μl of purecompound have been dissolved in 100 μl plasma at a temperature of 37° C.The resulting solution is stirred. After a 3 or 24 hour period of time,5 μl of internal standard (N-benzylamine) and 1 ml ether are added tothe solution maintained at 37° C. The sample is centrifuged for 10minutes at a rate of 13,000 rpm. 1 μl of the supernatant phase (etherphase) is injected in GC. The sample is then GC analyzed. Thechromatogram analysis allows to emphasize that after 3 hours in contactwith human plasma maintained at 37° C., the peak corresponding to thecompounds of the invention is still observed. The compounds of theinvention do not therefore undergo or undergo little significantdegradations after 3 hours in the presence of plasma. No other peakoptionally corresponding to metabolization products is observed. After24 hours in contact with human plasma, the presence of the peakcorresponding to the compound of example 1 is still observed. However,the peaks corresponding to the compound of example 2 are not found anymore.

Example 12 In vitro Effect on the Neutrophil Adherence to theEndothelial Cells being Incubated under Hypoxia

Endothelial cells have been incubated under hypoxia in the presence ofdifferent concentrations of the compounds of the invention. Afterincubation, the cells are co-incubated with a non stimulated humanneutrophil suspension. In normoxic conditions, the neutrophil adherenceto the endothelial cells is low (from 5 to 10%), but it increases byabout 3times under hypoxia.

The effect of the compound of example 1 on the neutrophil adherence tothe endothelial cells being incubated under hypoxia is characterized byan inhibition of the hypoxia induced adherence, and this depending onthe concentration between 10⁻⁵ mol/l and 10⁻⁸ mol/l. An activity optimumshould be obtained between 10⁻⁷ mol/l and 10⁻⁵ mol/l. Similarly, thecompound of the example 2 inhibits the hypoxia induced neutrophiladherence increase between 10⁻⁶ mol/l and 10⁻⁸ mol/l with a 71% maximumat 10⁻⁸ mol/l.

Example 13 In vivo Effect on Liver After Treatment of Rats Per os for 5Days

The rats have been treated per os either with solutions having anincreasing concentration of each of the compounds of the invention orwith DMSO-containing batches at the same concentration as that used fordissolving the molecules (control rats) at the rate of once a day for 5days.

Following the treatment, the rats are sacrificed. The mitochondria areisolated from the liver and their respiratory activity is measured every15 minutes for the 75 minutes following the isolation procedure.

The optimal concentration in pre-incubation for the compound of theexample 1 is 10⁻⁶ mol/l (0.236 μg/l). The dosis used to treat the ratsis therefore 2.36 mg/kg and 5 times inferior and 5 times higher and 20times higher doses have also been tested.

For the compound of the example 2, the optimal concentration inpre-incubation is 10⁻⁷ mol/l (0.0183 μg/ml). The tested doses aretherefore 0.18 mg/kg. As such dose is weakly active, 5 and 20 timeshigher doses have also been tested.

The compound of the example 1 increases in a dose dependent andsignificative way the hepatic mitochondria RCR after a 5 day treatmentwith a 25% increase at 2.36 mg/kg. At a higher dosis, the moleculebecomes weakly toxic and the RCR falls slightly below the controlvalues. Such a RCR increase is due to a clear decrease of respirationstage IV.

The compound of the example 2 increases in a dose dependent andsignificative way the mitochondria RCR with a 17% maximum at 3.6 mg/kg.This RCR increase is also due to a decrease of respiration stage IV.

Example 14 In vivo Effect on the Heart After Treatment of Rats Per osfor One Day

The rats have been treated per os with the 2.36 mg/kg concentrations ofthe compound of the example 1 or with the 3.6 mg/kg concentrations ofthe compound of the example 2, either with DMSO-containing batches atthe same concentration as that used for dissolving the molecules(control rats) at the rate of once.

The following day, the rats are sacrificed and their mitochondria areisolated from the heart. Their respiratory activity is measured every 15minutes for the 75 minutes following the isolation procedure.

The compound of the example 1 increases the heart mitochondria RCR aftera one-day treatment with a 41% increase at 2.36 mg/kg. The compound ofthe example 2 increases the mitochondria RCR after a one day treatmentwith a 56% increase at 3.6 mg/kg. Example 15

In vitro Application for the Apoptosis Treatment

Promyelocyte cells are incubated in the presence of apoptosis-inducingagents like the etoposide, a type II topoisomerase inhibitor. Afterincubation, different parameters indicative of the apoptosis aredetectable: caspase activation, cytochrome C release by themitochondria, DNA fragmentation, etc.

Unexpectedly, if the cells are incubated in the presence of etoposideand in the presence of the compound of the example 1, an apoptosisinduction inhibition is observed. Such an inhibition is a function ofthe dosis between 10⁻⁵ mol/l and 10⁻⁸ mol/l. Thus, caspase 3 activation,cytochrome C release by the mitochondria and DNA fragmentation areinhibited.

What is claimed is:
 1. A compound of formula I, a salt or a prodrugthereof corresponding to formula I:

wherein: R¹ is selected from the group consisting of CH₂, NH, and agroup wherein said group together with R³ and/or R⁴ forms an aromatic ornon-aromatic ring having 5 or 6 carbon atoms, optionally comprising oneach ring an heteroatom, preferably a nitrogen, oxygen or sulfur atom,R³ and/or R⁴ represent: an amine having the formula:

or a group having the formula —Z—R⁶ wherein: Z represents O or S, R⁵represents H or an alkyl group with 1 to 6 carbon atoms, R⁶ represents ahydrophobic group, preferably a terbutyl, an allyl or an aromatic ornon-aromatic ring having 5, 6 or 7 carbon atoms optionally comprisingone or several heteroatoms and wherein the carbon atoms are optionallysubstituted for by: an alkyl group R⁷ having 1 to 6 carbon, a ketonegroup, a hydroxyl group, an ester group, a halogen element (F, Cl, Br orI), a trifluoromethyl group (CF₃), or a group having the formula:

wherein: W represents CH or a nitrogen atom, Y represents S or O, and R⁸represents an alkyl group having 1 to 6 carbon atoms, preferably amethyl or ethyl group, with the additional condition that, if R³together with R¹ forms said above-mentioned ring or if R³ is either anamine of formula:

or the above-mentioned group of formula —Z—R⁶, R⁴ represents an alkylgroup having 1 to 10 carbon atoms, either saturated or unsaturated,optionally comprising one or more heteroatoms.
 2. A compound accordingto claim 1, characterized in that R⁵, R⁷ and R⁸ represent H or a methylgroup, and wherein R¹ is CH₂.
 3. A compound according to claim 1,wherein R¹ is CH₂ and in that R³ and R⁴ represent an amine having theformula:

or a group having the formula: —O—R⁶, wherein R⁶ represents a terbutyl,a cyclopentene, a cyclohexane or a benzene group the carbon atoms ofwhich are optionally substituted for by a methyl group, a ketone group,a hydroxyl group, an ester group, a halogen element or a trifluoromethylgroup (CF₃).
 4. A compound according to claim 1, wherein it is selectedfrom the group consisting of dicyclopenten-2-yl malonate, 2-propenyl3-oxa-2-oxobicylo[3.1.0]hexane-1-carboxylate, ethyl cyclopenten-2-ylmalonate, di-(S)-cyclopenten-2-yl malonate, methyl cyclopenten-2-ylmalonate, N,N′-diphenyl malonamide, ethyl N-phenylamido ethanoate, ethyl2,6-dimethyl-N-phenylamido ethanoate and ethylN-methyl-N-cyclohexylamido ethanoate, optionally their salts or prodrugsthereof.
 5. A pharmaceutical composition comprising an appropriatepharmaceutical carrier and one or more compounds according to claim 1.6. Method for preparing the compound according to claim 1, comprisingthe following steps: esterification of a R³—H formula intermediatecompound by a group

in the presence of dicyclohexyl carbodiimide (DCC), optionally followedby one or more cyclizing reactions in the presence of an oxidizingagent.
 7. A method for treating and/or preventing ischemia, pathologiesassociated with ischemia or pathologies associated with mitochondrialdeficiencies, comprising administering a compound according to claim 1to a mammal.
 8. A compound according to claim 2, wherein R¹ is CH₂ andin that R³ and R⁴ represent an amine having the formula:

or a group having the formula: —O—R^(6,) wherein R⁶ represents aterbutyl, a cyclopentene, a cyclohexane or a benzene group the carbonatoms of which are optionally substituted for by a methyl group, aketone group, a hydroxyl group, an ester group, a halogen element or atrifluoromethyl group (CF₃).
 9. A pharmaceutical composition comprisingan appropriate pharmaceutical carrier and one or more compoundsaccording to claim
 2. 10. A pharmaceutical composition comprising anappropriate pharmaceutical carrier and one or more compounds accordingto claim
 3. 11. A pharmaceutical composition comprising an appropriatepharmaceutical carrier and one or more compounds according to claim 4.12. A method for treating and/or preventing ischemia, pathologiesassociated with ischemia or pathologies associated with mitochondrialdeficiencies, comprising administering a compound according to claim 2to a mammal.
 13. A method for treating and/or preventing ischemia,pathologies associated with ischemia or pathologies associated withmitochondrial deficiencies, comprising administering a compoundaccording to claim 3 to a mammal.
 14. A method for treating and/orpreventing ischemia, pathologies associated with ischemia or pathologiesassociated with mitochondrial deficiencies, comprising administering acompound according to claim 4 to a mammal.
 15. A method for treatingand/or preventing ischemia, pathologies associated with ischemia orpathologies associated with mitochondrial deficiencies, comprisingadministering a pharmaceutical composition according to claim 5 to amammal.
 16. The compound of claim 1, wherein the group comprises acarbon atom.
 17. The compound of claim 1, wherein R⁵ is a methyl groupor ethyl group.
 18. The compound of claim 1, wherein R⁷ is a methylgroup or ethyl group.